1. Field of the Invention
This invention relates to methods for detecting and quantifying occult blood employing a peroxidase indicator.
2. Description of the Prior Art
Detection of occult blood in stool samples may lead to the early diagnosis of colon cancer. Colorectal cancer detected in asymptomatic patients is usually localized without involvement of lymph nodes. When detection is delayed until the symptomatic stage, lymph node involvement is found in approximately 40% of the cases. Those patients receiving the benefits of early diagnosis have a 30% better survival chance as compared to those patients who have a late diagnosis.
Hemoglobin, being a peroxidase, can be detected and quantified by means of a peroxidase indicator comprising a reduced or decolorized dye and hydrogen peroxide. Peroxidases catalyze the decomposition of peroxide to water and oxygen. The liberated acceptor oxygen oxidizes the reduced indicator dye, whereby the color of the oxidized state is developed. There is a direct relationship between color intensity developed and peroxide decomposition which is in turn related to the amount of peroxidase present in a sample. By measuring color intensity, as by measuring absorbance at the optional wavelength, quantification of a peroxidase is possible.
Thus, occult blood may be detected using colorimetric tests based on the reaction of hemoglobin with a peroxidase indicator, such as hydrogen peroxide and either o-tolidine or gun guaiac. Other reagents have been described, but the o-tolidine and guaiac tests are commercially available and have been used in detection of occult blood. Occult blood tests in common use include preparations of gum guaiac (City Chemical Corp., New York), dilute alcoholic solutions of guaiac (Tincture of guaiac; Harleco, Philadelphia), orthotolidine tablets (Hematest; Ames Co., Elkhart, Ind.), and a guaiac slide (Hemoccult; Smith, Kline and French Laboratories, Philadelphia). The reliability and sensitivity of these tests can be determined by measuring occult blood independently using .sup.51 Cr-labeled red blood cells.
Serious doubts have been raised about the reliability of some of these peroxidase indicator tests because of the high frequency of false-positives. Such misleading results lead to the unnecessary performance of additional expensive and uncomfortable diagnostic tests.
False-positives can be due to the presence in faces of peroxidases, other than hemoglobin, which can produce positive reactions. Plant peroxidases, chlorophyl and animal hemoglobin and myoglobin may survive digestion and produce a positive reaction. Intestinal bacteria may also produce peroxidases. Other false-positive results can occur because of the ingestion of certain foods, e.g., bananas, red meat, and iron.
False-positives are avoided by some of the currently employed peroxidase indicator tests at the expense of sensitivity. Diminished sensitivity to occult blood, though effective to reduce false-positives, results in false-negatives which delay diagnosis. False-negatives may also be due to the presence of Vitamin C in feces.
Erroneous results with present colorimetric peroxidase tests can also be caused by the pigment in feces which blends with the color of the peroxidase indicator dye. Further, reducing agents present in feces may interfere with the action of the peroxidase.
The commercially available peroxidase indicator preparations for detection of occult blood are all semi-quantitative. That is, they yield results that are expressed as 0, +, ++, etc. Quantification, such as is available with detection using .sup.51 Cr-labeled cells, is not possible. The semi-quantitative nature of the results appears to be the result of the action of an inhibitor of hemoglobin activity in stool. The inhibitor is effective at high dilutions of stool in water, and its effects are completely eliminated only at a dilution of 1:8,000. Blood is detected only when it is present in excess of the inhibitor demand. Since this may occur at blood levels where prognosis is poor, these inhibitors reduce sensitivity, as well as prevent quantitative measurement.
The presence of an inhibitor of the peroxidase reaction in urine also interferes with detection and precludes exact quantification of myoglobin or hemoglobin. As with stool, readings for the presence of blood are reported on a 0 to ++++ scale.
The effects of the inhibitor can be overcome by using an excess if substrate, either chromogen or peroxide. As high concentrations of peroxide reduce hemoglobin activity, excess dye may be added. Although this permits detection of hemoglobin in urine or stool, the variable amount of inhibitor present makes exact quantification impossible.
Therefore, a need exists for methods of detecting occult blood which are sensitive enough to reliably detect small amounts of hemoglobin while discriminating against other peroxidases. Further, a need exists for an accurate colorimetric test for quantitatively detecting occult blood, so that by following serial fecal samples, it is possible to determine whether the bleeding is adventitiously present or is increasing in volume.